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There is a problem in understanding how viral RNAs (flaviviruses, bunyaviruses, arenaviruses) are translated into proteins:  the models developed for cellular RNAs don't fit some viral RNAs.


To explain:  Open most any textbook of biochemistry or molecular biology, turn to the chapter on translation of metazoan messenger RNAs (mRNAs), and you will see a model similar to the one shown at left.  It is called the "closed circle model" of messenger RNA translation. The messenger RNA has a 5' cap structure, a 5' untranslated region, a coding region, a 3' untranslated region, and a 3' poly(A) tail, consisting of up to 150 uninterrupted adenosine residues. The 3' poly(A) tail is recognized and bound by poly(A) binding proteins. Other essential "helper" proteins called eukaryotic initiation factors (eIFs) facilitate ribosome binding, unwind secondary structure, and facilitate translation initiation at the AUG start codon.  

The opposite end of the mRNA, the 3' terminus, also participates in the initiation of protein biosynthesis.  Biochemical experiments provide strong evidence indicating that the messenger RNA is circularizedfor translation by protein-protein interactions between poly(A) binding protein bound to the poly(A) tail, and eIF4G bound to the upstream or 5' mRNA end. 

But there is a problem with this model.  Messenger RNAs from several virus families, including Flaviviridae, Arenaviridae, and Bunyaviridae families don't have a poly(A) tail.  Therefore, the closed circle model of messenger RNA translation shown in the figure above cannot be strictly true for these viral RNAs because they lack the polyI(A) tail, the binding site for poly(A) binding protein (PABP).  If there is no PABP binding site, it is not clear how the 3' mRNA terminus interacts with eIF4G to circularize the RNA as shown in the illustration above. One model is that PABP binds the 3' untranslated regions of non-polyadenylated RNAs to facilitate viral RNA translation.

The poly(A) tail of metazoan messenger RNAs is thought to facilitate translation by ribosomes and to help protect the mRNAs from nucleolytic degradation..... so on the face of it,  the absence of the poly(A) tail seems counterintuitive.


Our approach to this problem is to apply a transcriptome-wide analysis of messenger RNA translation in virus-infected cells.  The illustration above shows double ovals (ribosomes) that are moving down the green line (mRNA). RNAs that translate very efficiently have many ribosomes, while inefficiently translated mRNAs have few bound ribosomes.  

We are asking how many ribosomes are bound to every messenger RNA (the transcriptome) present in uninfected and flavivirus-infected cells. The process of messenger RNA translation is competitive; that is, some mRNAs have sequence and structural features that permit high-efficiency translation, while other sequences and structures are less favorable. By determining the numbers of ribosomes bound to cellular and viral RNAs, we can take a step toward understanding how the viral RNAs compare in translational efficiency to cellular RNAs.  One important cellular RNA is also non-polyadenylated--some histone mRNAs--and we are curious to examine cellular poly(A) RNA translation, histone mRNA translation, and viral mRNA translation.


A method we are using to attack this problem is called ribosome profiling. In the illustration at left, the ribosomes (double ovals) are covering a stretch of about 30 nucleotides of mRNA nucleotide sequence.  These bound ribosomes will therefore protect the 30-nucleotide region from nucleolytic degradation if ribonuclease is added. By recovering the protected fragments (ribosome footprints) and determining their nucleotide sequences by next generation sequence methods, we can determine how many ribosomes are present on the mRNA, and subsequently calculate messenger RNA translational efficiency (number of completed polypeptides produced per ribosome per messenger RNA molecule).

In the illustration at left , "polysomes" (mRNA with multiple ribosomes) are visible, and the ribosome footprints are the ribosome-protected sequences that escape degradation. The lower part of the figure shows a histogram of ribosome coverage on the mRNA.  

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